Although long-lived classical MBCs (CD19+/CD20+/CD21+/CD27+/CD10-) are gradually acquired in response to natural infection, exposure to P. falciparum also results in a large expansion of what we have termed atypical memory B cells (MBCs) (CD19+/CD20+/CD21-/CD27-/CD10-). At present, the function of atypical MBCs in malaria is not known nor are the factors that drive their differentiation. We recently carried out studies to establish the relationship between atypical MBC and classical MBCs and provided evidence that atypical MBCs and classical MBCs have undergone the same number of cell divisions and that the antibody V gene repertoires and somatic hypermutation rates are indistinguishable suggesting a common developmental history. We showed that atypical MBCs have lost two key adaptive immune cell functions, namely the ability to signal through the BCR and to differentiate into Ab secreting cells. Atypical MBCs express an array of inhibitory receptors and BCR signaling is blocked at the level of phosphorylation of one of the early kinases in the BCR signaling pathway (Syk) resulting in impaired B cell responses. Moreover, atypical MBCs are unable to respond to a variety of stimuli that triggered the differentiation of classical MBCs to differentiate into Ab secreting cells. This year we established a collaboration with Dr. Jun Zhu, an expert in RNAs as regulators of cellular differentiation, to carry out an RNA seq analysis of RNAs in naive B cells, atypical MBCs and classical MBCs. We have thus far obtained high quality RNA sequences and are in the process of analyzing these data. Our goal is to determine which RNAs are necessary for the differentiation of atypical MBCs with a view toward understanding their function. We have also completed a study to define the factors that drive the differentiation of human naive tonsil B cells to acquire the phenotype of atypical MBCs in vitro. We determine that key factors included: the form in which antigen was presented to B cells with presentation on membranes being most effective, and the presence of gamma IFN and the TLR9 agonist CpG. Provided with these stimuli naive B cells expressed a variety of genes associated with atypical MBCs including T-bet and FcRL5 and lost their ability to be triggered through the BCR. An important mechanism for immune evasion of P. falciparum is the rapid antigenic variation achieved through the var gene products, PfEMP1-proteins. Each Pf genome contains approximately 60 antigenically distinct var genes that are clonally expressed in iRBCs and it has been proposed that acquiring Abs to these explains in part, the slow acquisition of immunity in malaria. The extracellular domains of PfEMP1 is composed of several, up to 10, domains and can be grouped into three main groups, A, B and C, and two intermediate groups, B/A and B/C based on the type and number of domains they contain. The acquisition of PfEMP1-specific Ab is an important issue and consequently we analyzed individuals in our longitudinal cohorts for the acquisition of PfEMP1-specific Abs using a PfEMP1-luminex array designed in collaboration with Dr. Louis Miller in NIAID LMVR and Drs. Joseph Smith at Seattle Biomedical Institute and Thomas Lavstsen at University of Copenhagen, both var gene experts. The array contains 45 Cystein-rich interdomain regions of PfEMP1 groups A and B as well as several control P. falciparum proteins and vaccine proteins. We learned that PfEMP1-specific Abs are acquired in order during a child's lifetime in an endemic area beginning with the acquisition of Abs to Var A. We are continuing this analysis to determine how this acquisition compares with that of Abs to other P. falciparum proteins, how long lived PfEMP1-specific Abs are and whether the acquisition of PfEMP1-specific Abs correlates with immunity to uncomplicated disease. We will also evaluate the cross-reactivity of PfEMP1-specific Abs and their autoreactivity. We described the antibody V gene repertoire in response to malaria in infants and young children in Mali in collaboration with Dr. Ning Jiang, University of Texas. We used a sequencing method based on the use of molecular identifiers in combination with a clustering method that revealed more than 80% of the antibody diversity from as few as 1,000 B cells. We discovered high levels of somatic hypermutation in infants as young as three months old that gradually increased with age and stabilized in toddlers. Our results highlighted the vast potential antibody repertoire diversification in infants that had not been previously recognized and could have a profound impact on vaccination strategies in children. Over the last year we also explored the relationship between Pf infections and autoimmune disease. We described the regulation of B cells expressing the inherently autoreactive VH4-34 heavy chain (identified by the 9G4 monoclonal antibody) and 9G4+ plasma IgG in adults and children in our Malian cohort. The frequency of 9G4+ peripheral blood CD19+ B cells was similar in U.S. adults and African adults and children, approximately 4%. However, more 9G4+ B cells appeared in classical and atypical MBC compartments in African children and adults as compared to U.S. adults. The levels of 9G4+ IgG increased following acute febrile malaria, but did not increase with age as humoral immunity is acquired or correlate with protection from acute disease. This was the case even though a portion of 9G4+ IgGs were specific for malaria antigens and contained equivalent numbers of somatic hypermutations as compared to all other VHs. Taken together these results suggest that 9G4+ IgG may play a role in the control of acute febrile malaria, but its production may be regulated to insure that high levels of long-lived autoreactive 9G4+ IgGs are not acquired with age. To better characterize the sequence of events that contribute to cerebral malaria we have established a collaboration with Dr. Dorian McGavern to use two photon intravital imaging to view the cellular events that occur following infection with a mouse Plasmodium that results in cerebral disease. These studies provided strong evidence that the interaction of parasite-specific CD8+ T cells with brain endothelium is necessary for the pathology of cerebral malaria. These results led us to test a number of inhibitors of T cell metabolism as adjunctive therapy for cerebral malaria in collaboration with Dr. Jonathan Powell at Johns Hopkins. Remarkably we found that glutamine analogue, 6-diazo-5-oxo-L-norleucine (DON), rescued mice from CM, when administered late in the infection when mice already show neurological signs of the infection. At the time of treatment mice are suffering blood-brain barrier dysfunction, brain swelling and hemorrhaging accompanied by accumulation of parasite-specific CD8+ effector T cells and infected red blood cells in the brain and perturbation of brain metabolism. Remarkably, DON-treatment restored blood-brain barrier integrity, reduced brain swelling, decreased the function of activated effector CD8+ T cells in the brain and returned brain metabolites to uninfected levels. We have established a collaboration with Dr. Terrie Taylor, an expert in CM in children who heads a pediatric clinic in Malawi that treats children with cerebral disease, to describe the immune infiltrates into the brains of children who died of CM using multiplex immunohistochemistry analysis of brain sections. We have also established a collaboration with Dr. Dima Hammoud, an expert in the application of MRI and PET imaging to the study of brain infections to image brains of mice with CM. Our goal is to better understand the similarities and differences in the pathology associated with CM in children in Malawi and in mice.